Process for the production of derivatives of 9-polyhydroxyalkylisoalloxazines and products obtained



UNITED STATES PATENT OFFICE PROCESS FOR THE PRODUCTION OF DE- RIVATIVESF 9-POLYHYDROXYALKYL- i SOAIIELOXAZINES AND PRODUCTS OR- AIN n CasimirFunk, New York, and A Jay Merritt, Elmont, N. Y., assignors to U. S.Vitamin Corporation, New York, N. Y., a corporation of Delaware NoDrawing. Application July 29, 1949, Serial No. 107,628

13 Claims. (Cl. 260- 2113) The invention relates to acid esters of9-p0lyof riboflavin, that are of relatively high solubility,

; hydroxyalkyl-isoalloxazines and salts thereof, particularly in water.

1' and to a method for their preparation. More Another object of theinvention is the provision I particularly, it pertains to thepreparation of deof a process in accordance with which derivativesrivatives of 9-polyhydroxyalkyl-isoalloxazines of 5 of a9-polyhydroxyalkyl-isoalloxazine of inincreased water-solubility andincludes correlatcreased water-solubility may be produced by reedimprovements and discoveries whereby such action with aphosphorus-containing substance, derivatives may be produced with theobtention and a water-soluble vitamin whereby acid esters of substancesof enhanced value. of a 9-polyhydroxyalkylisoalloxazine with a It willbe understood that mention particularly 1O water-soluble vitamin areobtained. Also, salts of riboflavin throughout the specification is byof such acid esters which are even more readily way of illustration onlyand not limitative. Risoluble and more stable than the acid esters mayboflavin is one of the factors of the B-complex be prepared.

and is known to be the least soluble of this water- An additional objectof the invention is the soluble series of vitamins. Further, it isrecogprovision of a process for the preparation of ribonized that thetherapeutic use of riboflavin in soflavin derivatives which aredistinctly more sollution is decidedly restricted because of its W ublein water and other harmless solvents than solubility in solvents whichmay be physiologicalthe unreacted riboflavin. 1y utilized, e. g., water,aqueous ethyl alcohol, A still further object of the invention is theglycerol and the like. 99 provision of a composition of matter which isit Much Work has been done seeking to increase derivative of a9-polyhydroxyalky1-isoalloxazine the solubility of riboflavin eitherthrough the proand f rmed by h r c ion b we n a 9p l h duction ofderivatives thereof containing solud oxyalkyls a Compound S ec ebilizing groups, or through the employment of from the group consistingof water-soluble vitasolubilizers for the riboflavin. Inasmuch as themins and lower aliphatic amino acids and hysolubility of riboflavin inwater is slight, the addrated phosphorus oxychloride. ministrationthereof hypodermically requires a A more particular object of theinvention is the large volume of liquid. Riboflavin at C. has provisionas a composition of matter of a derivaa solubility in pure water ofabout 0.013% and tive of riboflavin formed by reaction between rihencethe administration of a 10 milligram dose boflavin, a water-solublevitamin and a hydrated of riboflavin at 20 C. would require theinjecreactive phosphorus composition capable of tion of about 78 cc. ofsolution. Further, the solforming phosphorus acid esters. ubility ofriboflavin in organic solvents as glyc- A specific object of theinvention is to provide erol, alcohol and the like is even more limitedas a composition of matter a derivative of riboand this impedes thepreparation of concentrated fiavin formed by reaction betweenriboflavin, a multi-vitamin liquid products in desired propornicotinicacid compound and phosphorus oxytions. Moreover, the slight solubilityof ribochloride, or hydrated phosphorus oxychloride. flavin has made itimpossible to obtain a suifi- Other objects of the invention win in partbe ciently high dosage in conjunction with more obvious and will in partappear hereinafter. soluble vitamins in a volume suitable for use, soThe invention accordingly comprises the severthat if a greater volumewere employed in order al steps, and the relation of one or more of suchto p v a pr p r d sa f r r flavin, th steps with respect to each of theothers, and the higher dosages of the more soluble vitamins compositionspossessing the features, properties would result. I and the relation ofconstituents which are exem- Furthermore, riboflavin may be adm splified in the following detailed disclosure, and as a solid in the formof tablets or capsules, but the scope of the invention will be indicatedin as it is sometimes desirable to administer it th l i pa y r inSolution f r l use, t ribO- In the practice of the invention,derivatives of flavin must be rendered more soluble and sufiia9-po1yhydroxyalkyl-isoalloxazine may be prociently so that atherapeutically efiective amount d e by process hi h comprises reactingmay be present in a reasonable amount 01 water mixture containing a9-.polyhydroxyalkyl-isoa1- or other inert or innocuous solvent.loxazine, and more particularly 6,7-dimethy1-9- An object of the presentinvention is to provide d-ribityl-isoalloxazine (riboflavin) and 6,7-diaprocess for the production of derivatives of amethyl-9-l-arabityl-isoalloxazine (arabofiavin),9-polyhydroxyalkyl-isoalloxazine, and especially a nicotinic compoundselected from the group of the 9-polyhydroxyalkylisoalloxazines and notby Way of limitation, the reaction mixture may contain riboflavin, anicotinicacid-compound and;

hydrated phosphorus oxychloride... As. pointed.

out above, the nicotinic compound maybe nicotinic acid (niacin),nicotinamide (niacinamide) or a salt of nicotinic acid as;thev calcium,sodium Furthermore, other waterand potassium salts.

soluble vitamins as ascorbic acidandpyridoxine may be employed, andlower aliphatic amino acids as glycine, alanine, beta-alanine and esters'WQEQE obtained when the hydrated phosphorus thereof may be utilized.

The proportions in which the various reactants may be employed may vary,with satisfactory results having been obtained with theiollowingrelative proportions: lnmole OfllbOr. flavin, 1 mole of anicotinic .compound, as nicor.v tini'c .acid; orni'cotinamide, and 15molesof hydratedlphosphorus oxychloride. These. DROPOI':

tions lead to the production of riboflavin deriv-.

atives which are markedly more soluble in water and. other harmlesssolventsthan is theunreacted. riboflavin.

As an illustrative embodiment of amanner inwhich .the invention maybepracticed, the

following examples. are presented. The. partsare by weight.

EXAMPLE 1 15.04-parts riboflavin. 4.88; parts niacinamide. 70.00 partshydrated phosphorus oxychloride.

In a suitable receptacle as a glass mortar of sufficient size grindthe..riboflavin and niacinamideuntil they are intimately mixed. Add,

the hydrated phosphorus oxychloride and mix intermittently over a periodof about 48.hours.. Add. 150,parts of cracked ice, stirring until alltheioehas melted, then transfer to a vessel desirably of pyrex glass forcrystallization, washing thereceptacleor mortar with two 10 cc. portionsof water.

much .ofthe mother liquor as possible; wash the product twice with 100ml. portions of. anhydrous ethyl alcohol; then wash twice with 100 ml.,portions of ethyl ether, and dry in vacuo.

The yield was 15.0 parts dry material which is. 67% of theoretical.Hence, the theoretical riboflavin content is 67.0%, and that foundequals 66.2% (fluorometric analysis). of the product afterrecrystallization gave. the following results:

This empirical formula indicates that the prod- I Allow to crystallizeovernight atabout 10? C Filter by suction, removing as.

Microanalysis ter under mixing and cooling. For example, to 1515;gramsoi phosphorus oxychloride (1 mole) areadded 3dgramsl2 moles) ofwater in a suitable glass vessel, dropping the water in slowly over aperiod of about two days with agitation at a temperature of.:abo ut 0C'. The mixture is -thenstir-redrintermittently for a period of abouttwo weeks unti-l the mix becomes viscous and most of the'hydr-ochloricacid which is formed in the reaction has been removed. Best results riedoutina suitable vessel, with mechanicalagitation; means for removing thehydrochloric acid gasiorrnedyin-the reaction, and means for cooling thereaction mixture, Further, the-reac tion may also becarried-outsothatthefmalproduct may be obtained in a periodof; less than 48;hours-.order to obtain-a purified-product free from excessiveamounts-of tarryby-productsit is important that the reaction; between--a hydroxy groupof the riboflavin and the phosphorie-- acid be slowed somewhat. It isbelievedthatthe-- reaction products are esters oithe vitamins andphosphoric acid,- and while the structure of the compositionhas not beendefinitely established,

neverthelessit is thought-to be as follows:

e lar? 0 15.-. 4..-. arts ibofla i .88.;p rts. ac namida;

70-00 P is y rats i hos e s e hl der a? ast wo eeks 19 Inasuitablereceptacle-orfvessel; as a glass mortar grind the riboflavinand niacinarnideuntil they are intimately mixed. Add-the hydratedphosphorus oxychloride andmix intermittentlylover a period of about {23hours. 'Add- 1'50parts of gcracked-ice, stirring until all the V r Thereaction mixtureis now treated with 28 augnmonia water until a pl-lofabout 5.5 is obtained. The mixture is thenice has melted.

shaken or agitated with an adsorbing powder such as a Norite charcoalfor -15 -to 20 minutes,

using about 7 to-10par tsof the charcoal per part of riboflavin: Thecharcoal 'oriotherladsorbate. is 'filte'redoffand Washed; e.-;g., in.aisoxhlet. ape.

paratus, with water until free of chloride and phosphate ions.

Elution of the product from the charcoal adsorbate is carried out by anazeotropic mixture of pyridine (57%) and water (43%). When the elutionis complete, the eluate is evaporated to dryness. The residue isdissolved in a minimum amount of boiling water and hot ethyl alcohol isadded until beginning of crystallization. The mixture is then cooled andallowed to stand in a cooled space for a period, as overnight, forcrystallization. The crystalline product is illtered, washed withanhydrous alcohol and ether and dried in a vacuum chamber. The yield was3.4 parts of a crystalline powder. Additional recovery can be obtainedby further evaporation of the mother liquors.

EXAMPLE '3 Preparation of the ammonium salt of the soluble riboflavincompound To prepare the ammonium salt of the soluble riboflavincompound, the free acid prepared as in Example 1 is mixed with water, 10ml. of water per gram of the riboflavin compound, and the pH brought to6.5-7.0 with 28% ammonia water. Insoluble material, if any, is removed,and anhydrous ethyl alcohol is added in an amount to give an alcoholconcentration of 30%. The mixture is then placed in a cool spaceovernight and the ammonium salt crystallizes. The salt is separated byfiltration, washed with ethyl alcohol, then with ether, and dried invacuo. Additional ethyl alcohol in an amount to give an alcoholconcentration of 50% is added to the mother liquor and a further amountof the ammonium salt crystallizes in the cold. After separation of thesecrystals, alcohol to give a concentration of 80% is added to the motherliquor and a third formation of crystals is obtained.

Starting with 4.0 grams of the riboflavin compound, described in Example1, a total of 3.4 grams of the ammonium salt is obtained. This compoundis soluble in water to the extent of about 50 mgms. per cc.

The foregoing procedures yield derivatives of riboflavin which arecharacterized by markedly increased solubility in water in comparisonwith riboflavin, such that by way of example solutions may be preparedcontaining from 12-l3 milligrams per milliliter of solution. Further,the

products as the derivatives of riboflavin may be looked upon somewhat assubstances containing '1 mole of riboflavin, 1 mole of a nicotiniccompound, as nicotinic acid, nicotinamide, and a salt of nicotinic acid,and a phosphoric acid group. Additionally, the derivatives have anaverage content of phosphorus from about 5% to about 6%. While theamounts of the reactants and the conditions under which the derivativesare formed desirably are those given in the foregoing examples,nevertheless, the amounts and the conditions may be varied somewhat, andsuch variations lead to the preparation of products of differentsolubilities. As set forth hereinbefore, watersoluble vitamins may beutilized as reactants, more particularly, nicotinic acid, nicotinamide,salts of nicotinic acid and ascorbic acid, and also that lower molecularweight saturated aliphatic amino acids as glycine may be employed inplace of the water-soluble vitamins. The products thus obtained possessthe property of increased water-solubility, stability in solution anddistinctive riboflavin activity as determined micro-biologically andfluorometrically.

' Since certain changes carrying out the above process, and certainmodifications in the compo-' sition which embody the invention may bemade without departing from its scope, it is intended that all mattercontained in the above description shall be interpreted as illustrativeand not in alirniting sense.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereinde-- scribed, and all statements of the scope of the invention which, asa matter of language, might be said to fall therebetween.

Having described our invention, what we claim as new and desire tosecure by Letters Patent is:

1. A process for the production of derivatives of a9-polyhydroxyalkyl-isoalloxazine which comprises reacting a mixturecontaining a 9-polyhydroxyalkyl-isoalloxazine, a nicotinic compoundselected from the group consisting of nicotinamide, nicotinic acid andthe calcium, sodium and potassium salts thereof, and hydrated phosphorusoxychloride.

2. A process for the production of derivatives of a9-polyhydroxyalkyl-isoalloxazine which comprises reacting a mixturecontaining a 9-polyhydroxyalkyl-isoalloxazine, a nicotinic compoundselected from the group consisting of nicotinamide, nicotinic acid andthe calcium, sodium and potassium salts thereof, and hydrated phosphorusoxychloride at a temperature at least as low as 10 C.

3. A process for the production of derivatives of a9-polyhydroxyalkyl-isoalloxazine which comprises reacting a mixturecontaining a 9-polyhydroxyalkyl-isoalloxazine, nicotinamide and hydratedphosphorus oxychloride riboflavin, a nicotinic compound selected fromthe group consisting of nicotinamide, nicotinic acid and the calcium,sodium and potassium salts.

4. A process for the production of derivatives of riboflavin whichcomprises reacting a mixture containing riboflavin, a nicotinic compoundselected from the group consisting of nicotinamide, nicotinic acid andthe calcium, sodium and potassium salts thereof, and hydrated phosphorusoxychloride at a temperature as low as 10 C.

5. A process for the production of derivatives containing riboflavin,nicotinamide, and hydrated phosphorus oxychloride, in the respectiveapproximate molar ratio of 1 1:15.

sium salts thereof, oxychloride, and having an average phosphoruscontent from about 5% to about 6% and a water-solubility greater thanthat of the unreacted 9-polyhydroxyalkyl-isoalloxazine.

9; As aoomposition oi mattersa riboflavin .derivative comprisingthereaotion 'productof riboe fiavin, a nicotinic compound selected ifrom the group: consisting of= nicotinamide, nicotinio acid and thecalcium, sodium: and potassium salts thereof, and. hydrated phosphorusoxycnloride, and having an average phosphorus content'from about 5% toabout 6% and'a waterz-solubility greater than that of theunreactedribofiavin.

10. As a composition of matter a riboflavin derivative. comprisin thereaction produotuof riboflavin and-nicotinamide in essentially equimolar proportion and hydrated phosphorus oxychioride, said derivativehaving an average phosphoruscontentfrom about 5% to about 6% and 15 aWater-solubility greaterthan that of riboflavin. 11. A' composition ofmatter as defined in claimsiin the form ofan ammonium salt.

12; A composition of matter as defined in ciaim-9the form=of an ammoniumsalt;

13; A composition of matter as defined in claim 10 'in the form of anammonium salt.

CASIlVIlRFUNK. AJAY MERRITT.

References Cited in the-file of this patent UNI E STATES PA EN SMeerwein et a1., Chem. Abst, v01. 24 (1930), page 1049.

Booher, Chem. and Ind., September 19, 1942,

7 page 390..

Gerrard et al., Chem. Abst, vol. 39 19.45) page.

1. A PROCESS FOR THE PRODUCTION OF DERIVATIVES OF A9-POLYHYDROXYALKYL-ISOALLOXAZINE WHICH COMPRISES REACTING A MIXTURECONTAINING A 9-POLYHYDROXYALKYL-ISOALLOXAZINE, A NICOTINIC COMPOUNDSELECTED FROM THE GROUP CONSISTING OF NICOTINAMIDE, NICOTINIC ACID ANDTHE CALCIUM, SODIUM AND POTASSIUM SALTS THEREOF, AND HYDRATED PHOSPHORUSOXYCHLORIDE.